1. Field of the Invention
The present invention relates generally to transgenic plants. More specifically, it relates to methods and compositions for transgene expression using a promoter naturally associated with a Zea mays nuclear gene encoding a cytoplasmic glutamine synthase.
2. Description of the Related Art
An important aspect in the production of genetically engineered crops is obtaining sufficient levels of transgene expression in the appropriate plant tissues, especially tissues that are involved in reproductive functions. In this respect, the selection of promoters for directing expression of a given transgene is crucial. Promoters which are useful for plant transgene expression include those that are inducible, viral, synthetic, constitutive as described (Paszkowski et al., 1984; Odell et al., 1985), temporally regulated, spatially regulated, and spatio-temporally regulated (Chau et al., 1989).
A number of plant promoters have been described with various expression characteristics. Examples of some constitutive promoters which have been described include the rice actin 1 (Wang et al., 1992; U.S. Pat. No. 5,641,876), CaMV 35S (Odell et al., 1985), CaMV 19S (Lawton et al., 1987), Ti plasmid nopaline synthase (nos, Ebert et al., 1987), alcohol dehydrogenase (Adh, Walker et al., 1987), and sucrose synthase (Yang and Russell, 1990).
Examples of tissue specific promoters which have been described include lectin (Vodkin et al., 1983; Lindstrom et al., 1990), corn alcohol dehydrogenase 1 (Vogel et al., 1989; Dennis et al., 1984), corn light harvesting complex (Simpson, 1986; Bansal et al., 1992), corn heat shock protein (Odell et al., 1985; Rochester et al., 1986), pea small subunit RuBP carboxylase (Poulsen et al., 1986; Cashmore et al., 1983), Ti plasmid mannopine synthase (Langridge et al., 1989), Ti plasmid nopaline synthase (Langridge et al., 1989), petunia chalcone isomerase (Van Tunen et al., 1988), bean glycine rich protein 1 (Keller et al., 1989), truncated CaMV 35s (Odell et al., 1985), potato patatin (Wenzler et al., 1989), root cell (Conkling et al., 1990), maize zein (Reina et al., 1990; Kriz et al., 1987; Wandelt and Feix, 1989; Langridge and Feix, 1983; Reina et al., 1990), globulin-1 (Belanger and Kriz et al., 1991), α-tubulin (Carpenter et al., 1992; Uribe et al., 1998), cab (Sullivan et al., 1989), PEPCase (Hudspeth and Grula, 1989), R gene complex-associated promoters (Chandler et al., 1989), chalcone synthase promoters (Franken et al., 1991) and glutamine synthetase promoters (U.S. Pat. No. 5,391,725; Edwards et al., 1990; Brears et al., 1991).
Inducible promoters which have been described include ABA- and turgor-inducible promoters, the promoter of the auxin-binding protein gene (Schwob et al., 1993), the UDP glucose flavonoid glycosyl-transferase gene promoter (Ralston et al., 1988); the MPI proteinase inhibitor promoter (Cordero et al., 1994), the glyceraldehyde-3-phosphate dehydrogenase gene promoter (Kohler et al., 1995; Quigley et al., 1989; Martinez et al., 1989) and a light inducible plastid glutamine synthetase gene from pea (U.S. Pat. No. 5,391,725; Edwards et al., 1990).
Promoters that are active in functions relating to the development of male or female reproductive tissues as well as seed specific activities have also been described. For example, promoters for anther-specific genes such as apg from Arabidopsis (Roberts et al., 1993) and ra8 from rice (Jeon et al., 1999), and pollen-specific genes such as lat52 and lat59 from tomato (Twell et al., 1990), and ZM13 from maize (Hamilton et al., 1992) have been disclosed. Promoters for genes involved in the development of female tissues such as the Msg gene from soybean (Stromvik et al., 1999) and the chalcone synthase A gene from petunia (van der Meer et al., 1990) have been reported. Regulatory sequences for genes involved in the development of embryos or endosperm have been disclosed, for example the Brassica napin storage protein promoter (NapA; Ellerstrom et al., 1996) and the Opaque2 promoter from maize (Gallusci et al., 1994). However, while a promoter may be expressed in a reproductive tissue, it may also show regulation in an unrelated tissue; for example the ADH1 promoter of maize was found to express in roots as well as in endosperm tissues (Kyozuka et al., 1994). Considering the complex regulation that occurs during the formation of reproductive organs in higher plants, relatively few promoters specifically directing this aspect of development have been identified. It would be of benefit to the art to increase the number and variety of promoters involved in the development of reproductive organs.
Glutamine synthetase (EC 6.3.1.2) plays a key role in nitrogen metabolism in a diverse array of organisms including bacteria, humans and plants. More specifically, the enzyme catalyzes the addition of ammonium to glutamate to synthesize glutamine in an ATP-dependent reaction. Bacterial forms of glutamine synthetase (GS) are well characterized, however, the enzyme has received less study in eukaryotes (reviewed in Eisenberg et al., 2000). Clones for several procaryotic and eukaryotic glutamine synthetase genes have been isolated. Despite significant overall differences at both the nucleotide and protein levels, enzymes from assorted species show highly conserved amino acid residues believed to be important for active site function. This conservation of select residues suggests that the various enzymes function via a similar catalytic mechanism (Eisenberg et al., 2000).
In higher plants, glutamine synthetase is found in a variety of tissues, including leaf, root, seed, root nodule and fruit. In addition, there are two forms of glutamine synthetase: a cytosolic form (GS1) typically found in roots and leaves, and a plastidic form, primarily found in leaves (GS2). The cytosolic form is further characterized as being present in several different isoforms, or isozymes, within a plant.
The various isoforms of glutamine synthetase function as members of a complex cycle in the plant, with roles including the detection of inorganic nitrogen sources, ammonium assimilation, incorporation of acquired nitrogen into organic forms and the reassimilation of nitrogen released during metabolism (reviewed in Coruzzi, 1991; McGrath and Coruzzi, 1991; Lam et al., 1996; Stitt, 1999; Oliveira et al, 2001). Thus, glutamine synthetase affects growth, development and overall plant metabolism, and especially carbon metabolism (see McGrath and Coruzzi, 1991; Lam et al., 1996; Stitt, 1999; Oliveira et al., 2001).
Several glutamine synthetase genes have been isolated and all have been found to be encoded by nuclear genes. The plastidic form of the enzyme appears to be coded for by a single gene while the various isoforms of the cytosolic enzymes are coded for by small, multigene families (Tingey et al., 1987; Sakamoto et al., 1989; Brears et al, 1991; Li et al., 1993; Dubois et al., 1996; Lam et al., 1996). The members of the multigene families are believed to encode different subunits which may combine to form homo- or hetero-octamers (Tingey et al., 1987; Dubois et al., 1996) and various octet formations may account for the multi-faceted roles glutamine synthetase plays in overall nitrogen metabolism.
Plastidic and cytoplasmic glutamine synthetase genes have been studied in a number of plants and show a variety of regulation patterns (Lam et al., 1996). Rice plants, which utilize a C3 carbon metabolism and are typically grown in water flooded soils, appear to express cytosolic forms of GS in the submerged root tissues. In the rice plant leaves, cytosolic forms are found in vascular tissues and a plastidic form shows light-regulated expression (see Tobin and Yamaya, 2001). In barley, which also utilizes a C3 carbon metabolism pathway but is grown in soil with good aeration, both plastid and cytosolic versions are found to be expressed in the root tissues (see Tobin and Yamaya, 2001). In pea, which fixes nitrogen via a symbiotic relationship with nitrogen fixing bacteria, only cytosolic forms were found in root tissues and, as found with other plants, the plastidic version is light regulated (Tingey et al., 1988).
Promoters have been isolated for a number of GS genes and have been found to be diverse in sequence and activity. For example, glutamine synthetase γ and β from French bean are both expressed in the roots, yet the two genes showed different spatial and temporal patterns of expression (Forde et al., 1989, 1990). Promoters for cytosolic (GS3A) and chloroplastic (GS2) from pea were isolated and joined to a reporter gene. The assay showed that the two promoters exhibited independent expression patterns which indicated non-redundant functions for these genes (Edwards et al., 1990).
The cereal crop maize (Zea mays or corn) utilizes a C4 type metabolism for managing carbon resources. Li et al., (1993) and Sakakibara et al., (1992) reported that a total of six different GS genes in maize showed five different patterns of transcript accumulation in a variety of plant tissues. Protein studies demonstrated that a pair of glutamine synthetase isozymes were expressed to high levels in kernels during development (Muhitch, 1988; 1989). Detailed studies with monoclonal antibodies distinguished between the isoforms and showed that while form GSp2 was found the pedicel and other tissues, form GSp1 was localized mainly to the pedicel (Muhitch et al., 1995), a tissue that joins the developing kernel to the cob and houses the vascular tissue that feeds the developing kernel. Rastogi et al., (1998) disclosed that the gene for pedicel specific GSp1 was a previously identified cytosolic gene, GS1-2. Earlier research had reported that GS1-2 RNA accumulated mainly in roots (Li et al., 1993), but contradictory studies showed no accumulation of this RNA in roots (Sakakibara et al., 1995). Later work demonstrated that GS1-2 RNA was present in pedicel tissues and increased in accumulation from at least 7 to at least 33 days after pollination (Rastogi et al., 1998).
Seed development in maize, and other crops, requires the transport and transfer of carbon, nitrogen and other nutrients from vegetative plant sources via the phloem, through the pedicel, to the seed, or kernel sink. Uptake of these nutrients is critical for proper kernel development, reducing kernel abortion, grain fill, grain quality and overall grain yield. Nitrogen is carried in the vascular sap in the form of amino acids, in particular, glutamine, glutamate, aspartate, alanine and serine (Lyznik et al., 1982; Muhitch, 1989; 1995) with glutamine being one of the most abundant (Lyznik, et al, 1982; Porter et al., 1987; Oliviera et al., 2001).
Nutrient molecules are unloaded from the pedicel vascular sap via parenchymal cells in the phloem and move through several layers of tissue including the pedicel-placento-chalazal region and the endosperm basal transfer cell layer as they cross from maternal tissue to developing endosperm and embryo (see, for example, Kiesselbach and Walker, 1952; Lyznik et al., 1982; Thorne, 1985; Muhitch, 1993). The role of these tissues in metabolite transfer is very important as the developing kernel lacks vascular tissue of its own. Glutamine synthetase in the pedicel region is neglible very early after pollination, increases beginning about 10 days post-pollination (Muhitch, 1988) and activity increases with kernel development. Maximum glutamine synthetase activity, observed around 28 days post pollination, is coincident with maximum nitrogen assimilation into the kernel followed by a decrease in activity as the kernel matures to completion (Muhitch, 1988; 1989). As the kernel increases in size and matures, movement of nutrient molecules into the kernel decreases significantly, and certain transfer tissue are eventually crushed, effectively sealing off the mature kernel from the parent plant tissue (Kiesselbach and Walker, 1952).
Although the above studies have provided a number of useful tools for the generation of transgenic plants, there is still a great need in the art for novel promoter sequences with beneficial expression characteristics, particularly for promoters which are developmentally regulated in tissues which affect kernel development. The number of effective promoters available for use with transgenes in maize is not abundant and those specific to kernel development even smaller. It would be especially advantageous to identify a promoter which plays a role in the import of nutrients into a developing seed as manipulations with such a promoter may allow for improvements to kernel development, grain yield, grain quality, pest resistance, stress resistance, fertility or decreased kernel abortion.
New promoters, such as that of the present invention, and especially promoters that will express differentially in maize female reproductive tissues, are useful. Such expression specific promoters are useful in minimizing yield drag and other potential adverse physiological effects on maize growth and development that might be encountered by high-level, non-inducible, constitutive expression of a transgenic protein in a plant. A wider range of effective promoters also may make it possible to introduce multiple transgenes into a plant, each fused to a different promoter, thereby minimizing the risk of DNA sequence homology dependent transgene inactivation (co-suppression). Therefore, there is a great need in the art for the identification of novel developmentally regulated, reproductive tissue specific promoters which can be used for the high-level expression of selected transgenes in economically important crop plants.